The present invention relates generally to polymeric sensing membranes, and more particularly to polymeric sensing membranes comprising luminescent dyes and polymeric matrices having predictable gas permeabilities, and methods of making these membranes.
The ability to monitor gas concentration is advantageous in a variety of situations. For example, reactions carried out on a large scale in the chemical industry, such as fermentation reactions, often require the measurement of certain reactant or product gases. In medical care, continuous monitoring of the respiratory gases is becoming a common procedure for the study of respiration, assisting in anesthesiology and the treatment and diagnosis of cardiopulmonary disorders. In particular, it is often desirable to be able to monitor the level of oxygen in blood using in vitro methods.
One approach to measuring oxygen levels in blood is to use an oxygen-sensitive luminescent membrane. Such membranes typically comprise a polymeric matrix material and luminescent dye molecules dispersed within the polymeric material. The luminescent molecules are capable of emitting fluorescence or phosphorescence from excited electronic states which can be collisionally quenched by molecular oxygen. This process is commonly known as Stern-Volmer quenching and is described by the relationship EQU F.sub.0 /F=.tau..sub.0 /.tau.=1+.tau..sub.0.multidot.k.sub.q [0.sub.2 ] (1)
where F.sub.0 and .tau..sub.0 are the luminous intensity and the relaxation time for the luminescent dye in the absence of molecular oxygen, F and .tau. are the luminous intensity and the relaxation time when the molecular oxygen concentration [O.sub.2 ] is greater than zero, and k.sub.q is the quenching constant for the luminescent dye molecule. This equation is often rewritten in the form EQU F.sub.0 /F=.tau..sub.0 /.tau.=1+k.sub.SV.multidot..rho.O.sub.2 (2)
where k.sub.SV is the Stem-Volmer constant (in (mmHg).sup.-1 or torr.sup.-1) and .rho.O.sub.2 is the partial pressure of oxygen. For the application described here, partial pressures are given in units of mmHg where one mmHg is equivalent to one torr.
In use, oxygen-sensitive membranes are exposed to electromagnetic radiation capable of exciting the luminescent dye molecule from the ground electronic state to an excited electronic state. This usually involves the excited singlet state for fluorescent molecules or the longer lived triplet state for phosphorescent dye molecules. When the luminescent dye molecules undergo a transition from the excited electronic state back to the ground state, a photon is emitted at a characteristic wavelength. The amount of oxygen in blood can be determined by measuring a change in the luminescent state of dye molecules, since the decay rate from the excited state is altered by the presence or absence of oxygen gas.
Several different techniques exist which are designed to measure the emission properties of luminescent dye molecules dispersed within a matrix or polymeric materials. For example, one can adapt a relatively simple approach of measuring the fluorescence intensity elicited by a constant excitation source, e.g., as disclosed in U.S. Pat. No. 4,476,870. U.S. Pat. Nos. 4,810,655 and 4,895,156 further disclose methods of measuring the time resolved emission of a luminescent dye molecule dispersed within a polymeric material. If a pulse of light, used to excite a dye molecule, is of a relatively short duration (t&lt;&lt;.tau.), the decay of emission intensity from the initial value F.sub.i will be approximately described by EQU F(t)=F.sub.i.multidot.e.sup.-t/.tau. (3)
Various sampling and regression schemes can be used to estimate a value for .tau..
As disclosed in U.S. Pat. Nos. 5,127,405, 5,281,825, and WO Application No. 92/19957, measurement of the phase shift for luminescence emitted by a luminescent dye molecule may be accomplished using a modulated excitation source. The excitation signal can be modulated such that the source intensity varies sinusoidally ##EQU1##
where E.sub.p is the peak excitation source intensity and .omega. is the angular frequency of the excitation signal. The luminescence emission signal F(t) from the dye will also vary sinusoidally at the same frequency as the excitation signal, except with a phase lag which is related to the relaxation time by ##EQU2##
where F.sub.p is the luminescence intensity that would result from a constant excitation at intensity E.sub.p and .PHI. is the phase lag. The relaxation time can be calculated from the observed phase lag using the equation ##EQU3##
Also, as disclosed in, e.g., U.S. Pat. No. 4,716,363 and WO application No. 90/07107, a constant phase method for extracting relaxation information from samples may be employed through the use of a variable frequency modulated source. This constant phase technique includes adjusting the frequency (f) of the modulated excitation source with a feedback loop so that a constant phase lag, preferably in the range of 45.degree., is maintained. A rearrangement of equation (6) and substitution of .omega.=2.pi.f yields: EQU f=tan .phi./(2.pi..tau.) (7)
If .phi. is held constant at 45.degree., then tan .phi.=1, and by substituting .tau. from equation (2), it can be shown that: EQU f=(1+k.sub.SV.multidot..rho.O.sub.2)/2.pi..tau..sub.o (8)
Thus, the operating frequency of the feedback loop is directly proportional to the partial pressure of oxygen; as such, the constant phase method provides several advantages over the time resolved and constant frequency methods. For example, the constant phase method significantly reduces the complexity of the calculations required to produce a reported result. In addition, maintaining a constant phase lag permits the phase detector to operate in the most sensitive part of the response curve and optimizes signal to noise ratios by maintaining a constant signal amplitude over a wide range of oxygen partial pressures.
The time resolved, phase shift or frequency modulation methods are all advantageous particularly for the measurement of samples such as blood or milk; the light scattering characteristic of these samples will not affect the apparent quenching constants for the luminescence. However, while these methods may eliminate the need for optically opaque cover membranes to reduce optical interference of simple fluorescence amplitude-based measurements (such as disclosed in U.S. Pat. Nos. 4,919,891; 5,081,041, and 5,091,800), the instrumentation needed to perform such relaxation-based measurements is decidedly more complex.
Different luminescent dye/polymeric material combinations have been employed to make oxygen sensing membranes. For example, U.S. Pat. Nos. 4,003,707 and 4,476,870 disclose the use of near-UV absorbing dyes from the pyrene and pyrelene families, respectively. U.S. Pat. Nos. 4,587,101; 4,752,115; 5,030,420, and 5,631,340 advocate the use of ruthenium complexes which undergo Stern-Volmer quenching by oxygen and have longer lived excited states, as outlined in Anal. Chem. 63,337 (1991). Lanthanide complexes, which also have longer lifetimes, have also been used for oxygen sensing purposes, e.g., U.S. Pat. No. 4,861,727. Other luminescent dye molecules, such as porphyrin derivatives, are disclosed in U.S. Pat. No. 5,043,286 and WO application No. 95/10522, as well as in Biosensors and Bioelectronics 7, 199 (1991), Anal. Chem. 67, 4112 (1995), and J. Chem. Soc. Perkin Trans. 2, 103 (1995).
Polymeric matrix materials which have been used include those disclosed in U.S. Pat. Nos. 4,587,101; 4,752,115 and 5,043,286, teaching that unplasticized polymers or untreated sol-gels (e.g., U.S. Pat. No. 5,047,350) offer relatively poor performance when used in dye-based gas sensing membranes. At the same time, plasticized membranes or additives are likewise disadvantageous, since plasticizers and additives can leach out and affect oxygen permeability over time and under various storage conditions. U.S. Pat. No. 4,476,870 further discloses that oxygen sensing membranes having low oxygen permeable membranes are relatively insensitive. One response to poor performance or undesirable Stern-Volmer response is shown in U.S. Pat. No. 5,462,879, wherein a second dye with a different quenching constant was added to the polymer/dye membrane formulation.
Previous teachings regarding selecting appropriate polymeric materials and luminescent dye molecules for particular sensing membrane applications are sparse, and there has been only limited success in producing a reliable membrane having the specific response characteristic desired. As such, sensing membranes have been made with available polymeric materials and luminescent dye molecules using a more or less empirical or trial-and-error method, i.e., polymeric sensing membranes incorporating dye molecules having a known relaxation time .tau. are made, then tested to see if a membrane having the desired properties (Stern-Volmer response or k.sub.SV) was obtained. Thus, the sensitivity of a particular polymeric sensing membrane for a given combination of materials has only been determined after the fact. If the desired performance was not obtained, the practice of adding high amounts of plasticizers, (as taught in U.S. Pat. Nos. 4,587,101; 4,752,115 and 5,043,286), was performed to adjust the membrane properties. As a result, known polymeric sensing membranes require relatively complex and expensive equipment, offer relatively poor sensitivity or both.
In co-pending and commonly owned U.S. patent application Ser. No. 08/617,714, the entire disclosure of which is incorporated herein by reference, methods for choosing a dye and polymer combination which gives a desired response over a given dynamic range of .rho.O.sub.2 values are disclosed, and properties of dyes and polymers or heteropolymers which, when combined, result in membranes having desired oxygen sensing properties, are also disclosed. The method is based on experimental support for the finding that the Stern-Volmer constant of a polymeric sensing membrane is mathematically related to the relaxation time of the luminescent dye molecule and the oxygen permeability of the polymeric material in which the dye is dispersed. In particular, the Stern-Volmer constant may be given as: EQU k.sub.SV =4.pi..multidot.N.sub.A.multidot.P.multidot..tau..sub.o.multidot.Perm.sub. O2 (9)
where N.sub.A is Avogadro's number, p is the relative likelihood of an oxygen molecule colliding with a dye in an electronically excited state and Perm.sub.O2 is the permeability of the polymeric material. However, predictably determining suitable polymers having a desired range of O.sub.2 permeabilities for incorporation in oxygen sensing membranes heretofore has been lacking.